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Abstract:

The present invention relates to a biocompatible cationic nanogel
comprising a polymer network, said polymer network comprising polymer
units interconnected with one another through a cross-linking agent,
wherein said polymer network can be obtained by polymerizing
N-vinylcaprolactam and a cross-linking agent in a dispersed medium, in
the presence of a cationic initiator and a cationic or non-ionic
emulsifier. The invention also relates to methods for obtaining the
mentioned nanogels as well as to pharmaceutical compositions comprising
them.

Claims:

1. A biocompatible cationic nanogel comprising a polymer network, said
polymer network comprising polymer units interconnected with one another
through a cross-linking agent, wherein said polymer network is obtainable
by polymerizing N-vinylcaprolactam and a cross-linking agent in a
dispersed medium, in the presence of a cationic initiator and a cationic
or non-ionic emulsifier.

2. The nanogel according to claim 1, wherein said cross-linking agent is
a difunctional monomer comprising at least two vinyl groups.

4. The nanogel according to claim 1, wherein the cationic initiator is
selected from the group consisting of 2,2-azobis(N,N'-dimethylene
isobutyramidine)dihydrochloride (ADIBA),
2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate,
2,2'-azobisisobutyramidine dihydrochloride (AIBA),
2,2'-azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane}dihydrochlori-
de and 2,2'-azobis(1-imino-1-pyrrolidino-2-ethylpropane)dihydrochloride.

5. The nanogel according to claim 4, wherein the cationic initiator is
2,2-azobis(N,N'-dimethylene isobutyramidine)dihydrochloride (ADIBA).

6. The nanogel according to claim 1, wherein the emulsifier is a cationic
emulsifier.

7. The nanogel according to claim 6, wherein the cationic emulsifier is
selected from hexadecyltrimethylammonium bromide,
dodecyltrimethylammonium bromide and a quaternary ammonium block
copolymer.

8. The nanogel according to claim 7, wherein the quaternary ammonium
block copolymer is a styrene and 2-(dimethylamino)ethyl methacrylate
block copolymer of formula: ##STR00007## wherein n and m are integers
comprised between 5 and 50.

9. The nanogel according to claim 1, further comprising a biologically
active agent.

10. The nanogel according to claim 1 in the form of nanoparticles.

11. The nanogel according to claim 1 in a lyophilized form.

12. A pharmaceutical composition comprising a biocompatible cationic
nanogel as defined in claim 1, and a pharmaceutically acceptable
excipient, vehicle or adjuvant.

13. A method for obtaining a biocompatible cationic nanogel as defined in
claim 1, which comprises polymerizing a composition comprising
N-vinylcaprolactam and a cross-linking agent in a dispersed medium, in
the presence of a cationic initiator and a cationic or non-ionic
emulsifier.

14. The method according to claim 13, wherein said polymerization is
carried out in the presence of a biologically active agent.

15. The method according to claim 13, which further comprises contacting
said biocompatible nanogel with a solution comprising a biologically
active agent.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to thermo-sensitive, biocompatible
cationic nanogels as well as to a method for their production and to
pharmaceutical compositions comprising said nanogels. Among other
applications, the nanogels of the invention can be used as vehicles for
carrying and releasing biologically active agents.

BACKGROUND OF THE INVENTION

[0002] Over the last few decades, aqueous polymer dispersions prepared by
means of polymerization processes in a dispersed medium leading to the
production of polymer particles with sizes comprised within the colloidal
range have gained an increasing interest from both the academic and
industrial points of view (Forcada J. and Hidalgo, R., Curr. Org. Chem.
2005, 9, 1067-1084). These nanoparticles are used in a large variety of
applications ranging from adhesives, coatings based on water, paper,
textile, additives, flocculants, etc. They are also suitable as fine
polymer material or polymer material with high added value for medical
diagnostic tests, antibody purification, drug release systems and as
calibration material.

[0003] Nanogels are one particular type of these colloidal systems.
Nanogels are cross-linked colloidal particles which may swell by
absorbing large amounts of solvent, but they do not dissolve due to the
structure of the physically or chemically cross-linked polymer network
forming them.

[0004] Nanogels exhibit a behavior ranging from that of a polymer solution
(swollen state) to that of a "hard" particle (collapsed state). They can
respond to physical stimuli (temperature, ionic strength, magnetic
fields, electric fields, etc.), chemical stimuli (pH, ions and specific
molecules, etc.) and biochemical stimuli (enzyme substrates, affinity
ligands, etc.). Among the foregoing, temperature is the most widely
studied since it is a very effective stimulus in many applications. The
nanogels capable of undergoing a volumetric phase change upon changing
the temperature of the medium in which they are dispersed, or temperature
sensitive nanogels, are very interesting in those biotechnological
applications in which an active ingredient, a molecule or a material is
to be dosed in media in which the main variable is temperature.
Furthermore, it has been found that nanogel particles are capable of
responding to stimuli faster than their macroscopic gel counterparts,
i.e., hydrogels, due to their small size [Couvreur P. et al., Polymeric
Nanoparticles and Microspheres, Guiot, P.; Couvreur, P., ed., CRC Press,
Boca Raton, Fla., 27-93, 1986]. Therefore, the size of the particles
forming the nanogel is a very important parameter since it controls the
release system efficacy.

[0005] Nanogels used as vehicles for delivering and releasing molecules or
biologically active material must remain chemically unaltered and be
pharmacologically stable during their transit from the administration
site to the target where they are going to exert their effect. Likewise,
the characteristics of the vehicle must be such that it has to be not
only compatible with the path that it has to go through, but also with
the dosing site, i.e., it has to be "intelligent" and must release the
active ingredient at the precise moment when it has reached the target
and allowing the bioavailability thereof. On the other hand, the carrier
or vehicle must not only maintain its characteristics and properties in
an aqueous medium but must also be capable of showing its properties
again if it needs to be stored in a dry (lyophilized) state for the
application and/or transport. In other words, once redispersed, it must
again show its properties and behavior with respect to temperature
changes.

[0006] The most commonly used family of polymers in the synthesis of
microgels and nanogels sensitive to the conditions of the medium is
temperature sensitive poly(alkylacrylamides), more specifically
poly(N-isopropylacrylamide) (PNIPAM). However, the toxicity of
poly(alkylacrylamides) prevents their use in biomedical applications.
Despite this, there are many articles and patent documents published
throughout recent years regarding such systems. Cationic nanogels have
particularly been described. In this sense, Moselhy, J. et al. (Int. J.
Nanomedicine 2009, 4, 153-164) describe thermo-sensitive cationic
nanogels based on a polymer network of N-isopropylacrylamide (NIPAM) or
on a copolymer thereof with 2-(dimethylamino)ethyl methacrylate (DMAEMA)
and their potential use as DNA release vehicles; Eke, I. et al. (Colloids
and Surfaces A: Physicochem. Eng. Aspects 2006, 279, 247-253) describe
both pH and temperature sensitive cationic microgels synthesized from
N-isopropylacrylamide and N-3-dimethyl-aminopropylmethacrylamide (DMAPM)
and their use in the development of new surfaces related to
biotechnological applications; WO 2006/102762 proposes a technique for
grafting boronic acid groups into PNIPAM microgels with carboxyl groups,
which are used for the controlled release of insulin; Nolan, C. M. et al.
(Biomacromolecules 2006, 7(11), 2918-2922) also investigate the release
of insulin from PNIPAM microgels using variable-temperature 1H NMR;
Sahiner, N. et al. (Colloid Polym. Sci. 2006, 284, 1121-1129) refer to
obtaining pH sensitive cationic micro- and nanogels by means of
polymerizing (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) and
their numerous applications within the field of biomedicine and
biotechnology, particularly their use as vehicles for the controlled
release of active molecules.

[0007] Temperature sensitive biocompatible monomers include
N-vinylcaprolactam (VCL) [Vihola, H. et al. Biomaterials 2005, 26,
3055-3064], which is a water soluble monomer. Its corresponding polymer,
poly N-vinylcaprolactam (PVCL), combines very useful and important
properties because, in addition to being biocompatible, it has a phase
transition in the physiological temperature (32-38° C.) region.
This combination of properties allows considering it as a material
suitable for designing biomedical devices and for use in drug release
systems. Therefore, document WO 2008/145612, for example, describes
obtaining anionic N-vinylcaprolactam microgels suitable as an active
ingredient release system undergoing a progressive volumetric phase
change from the swollen state to the collapsed state as the temperature
of the medium in which they are dispersed increases.

[0008] Nevertheless, none of the cationic or anionic nanogels described in
the state of the art shows an immediate response to a temperature change
in the phase transition region, let alone said response involving a
volumetric change such that the nanogel changes from the collapsed state
to the swollen state as the temperature increases.

BRIEF DESCRIPTION OF THE INVENTION

[0009] The authors of the present invention have observed that a cationic
nanogel made up of a cross-linked polymer network of N-vinylcaprolactam
monomer units, obtained by polymerization in a dispersed medium in the
presence of a cationic or non-ionic emulsifier and a cationic initiator,
undergoes an instantaneous volumetric change at the phase transition
temperature of the polymer (32-38° C.), changing from the
collapsed state to the swollen state as the temperature increases, which
allows it to immediately release an active ingredient within the
physiological temperature region.

[0010] Therefore, in a first aspect, the invention relates to a
biocompatible cationic nanogel comprising a polymer network, said polymer
network comprising polymer units interconnected with one another through
a cross-linking agent, wherein said polymer network can be obtained by
polymerizing N-vinylcaprolactam and a cross-linking agent in a dispersed
medium, in the presence of a cationic initiator and a cationic or
non-ionic emulsifier.

[0011] An additional aspect of the present invention comprises a
pharmaceutical composition comprising a biocompatible cationic nanogel as
defined above, and a pharmaceutically acceptable excipient, vehicle or
adjuvant.

[0012] The last aspect of the present invention relates to a method for
obtaining a biocompatible cationic nanogel as defined above, which
comprises polymerizing a composition comprising N-vinylcaprolactam and a
cross-linking agent in a dispersed medium, in the presence of a cationic
initiator and a cationic or non-ionic emulsifier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows the transmission electron microscopy images taken at
approximately 22° C. and 60° C. clearly showing that the
nanogels of the invention are collapsed at temperatures below the phase
transition temperature of the polymer and swollen at temperatures above
said temperature.

[0014] FIG. 2 is a graph showing the variation of the average hydrodynamic
diameter with respect to the temperature experienced by the nanogels made
up of an N-vinylcaprolactam (VCL) and styrene (St) copolymer [a) 90%
VCL/10% St; b) 70% VCL/30% St; c) 50% VCL/50% St]

[0015] FIG. 3 is a graph showing the variation of the average hydrodynamic
diameter with respect to the temperature experienced by nanogels made up
of an N-vinylcaprolactam (VCL) and methyl methacrylate (MMA) copolymer
[a) 90% VCL/10% MMA; b) 80% VCL/20% MMA; c) 70% VCL/30% MMA; d) 50%
VCL/50% MMA].

[0016] FIG. 4 is a graph showing the variation of the average hydrodynamic
diameter with respect to the temperature experienced by nanogels of the
invention for different concentrations of emulsifier S7A13.

[0017] FIG. 5 is a graph showing the variation of the average hydrodynamic
diameter with respect to the temperature experienced by nanogels of the
invention for different concentrations of initiator: a) 1% by weight of
initiator ADIBA (E20I1); b) 0.5% by weight of initiator ADIBA (E20I0.5);
c) 0.3% by weight of initiator ADIBA (E20I0.3).

[0018] FIG. 6 is a graph showing the variation of the average hydrodynamic
diameter with respect to the temperature experienced by nanogels of the
invention with different emulsifiers: a) S7A13 (E20); b) HDTAB (H20); c)
DTAB (D20); d) Tween 20 (NI20).

[0019] FIG. 7 is a graph showing the variation of the average hydrodynamic
diameter with respect to the temperature experienced by the cationic
nanogel E20 after its lyophilization and resuspension.

DETAILED DESCRIPTION OF THE INVENTION

[0020] In one aspect, the invention relates to a biocompatible cationic
nanogel, hereinafter nanogel of the invention, comprising a polymer
network, said polymer network comprising polymer units interconnected
with one another through a cross-linking agent, wherein said polymer
network can be obtained by polymerizing N-vinylcaprolactam and a
cross-linking agent in a dispersed medium, in the presence of a cationic
initiator and a cationic or non-ionic emulsifier.

[0021] As used herein, the term "nanogel" refers to a three-dimensional
hydrogel particle the mean diameter of which is less than 1 μm, i.e.,
a diameter comprised between 1 and 999 nm. Likewise, a "hydrogel" is a
three-dimensional macromolecular network which swells in water and is
formed from a cross-linked polymer (e.g., polymer units interconnected
with one another by means of a cross-linking agent).

[0022] The cationic nature of the nanogel of the invention results from
using a cationic initiator in the polymerization process.

[0023] If desired, said nanogel of the invention can further contain at
least one biologically active agent, giving rise to a biocompatible
nanogel loaded with a biologically active agent which can be used for
carrying, delivering and/or dosing biologically active agents to the site
of interest. The biocompatible nanogels loaded or not loaded with
biologically active agents provided by this invention are sensitive to
the temperature changes of the medium in which they are dispersed, and
they can swell and shrink (i.e., change size), thus responding to
temperature stimuli.

[0024] In particular, the main advantage of the thermo-sensitive,
biocompatible cationic nanogels of the present invention with respect to
other nanogels of the state of the art is that in addition to being
biocompatible, they undergo an instantaneous volumetric change at the
phase transition temperature of the polymer, changing from the collapsed
state to the swollen state as the temperature increases, thus allowing
the immediate release of the active ingredient within the physiological
temperature region.

[0025] FIG. 1 shows the transmission electron microscopy images for a
cationic nanogel according to the present invention taken at
approximately 22° C. and 60° C., in which it can be
observed how the nanogel particles are swollen at temperatures higher
than the phase transition temperature of the main polymer making up the
polymer network and collapsed below said temperature. It furthermore
shows that the nanogel does not increase in size as a result of the
agglomeration or aggregation of several particles but rather as a result
of the swelling of the individual nanogel particles.

[0026] The biocompatible nanogel nanoparticles loaded or not loaded with a
biologically active agent provided by this invention can be lyophilized
and then resuspended in an aqueous medium, preserving their properties
and thermo-sensitivity. Therefore, the nanogels provided by this
invention are biocompatible materials, useful as carriers for
biologically active agents, capable of absorbing (encapsulating/uptake)
both biologically active hydrophobic and hydrophilic agents and suitably
dosing them since they respond to temperature stimuli of the medium in
which they are dispersed.

N-vinylcaprolactam (VCL)

[0027] N-vinylcaprolactam (VCL) is a compound with the following chemical
formula

##STR00001##

[0028] VCL is an amphiphilic monomer since it contains hydrophilic groups
(amide) and hydrophobic groups (vinyl group and alkyl groups of the
lactam ring). VCL is partially water soluble, its solubility being 8.5%
by weight. The water solubility of the corresponding homopolymer,
poly(N-vinylcaprolactam) (PVCL) varies with temperature. The presence of
the hydrophobic and hydrophilic groups means that repulsive and
attractive forces co-exist. The balance of these forces determines the
polymer solubility.

[0029] Some polymers are water soluble at low temperature, being separated
from the solution and forming a separate phase as the temperature
increases above a value known as "lower critical solution temperature"
(LCST), also called "phase change critical temperature". In the case of
PVCL, this phase change critical temperature is in the range comprised
between 32 and 38° C., i.e., close to the physiological
temperature.

[0030] PVCL further has an additional characteristic, its
biocompatibility, making it susceptible to being used in biomedical
applications. During the development of this invention, it has been
observed that the synthesis of said polymer must not be carried out at
acid pH for the purpose of preventing VCL hydrolysis.

[0031] The authors of the present invention have observed that the
copolymerization of VCL with other monomers causes a significant change
in the behavior of the polymer with respect to temperature. Therefore,
when VCL is copolymerized with styrene in a ratio of 50:50, the
nanoparticles are not temperature sensitive and behave like conventional
latex (see FIG. 2). Copolymerization with methyl methacrylate (MMA) in
turn provides particles which behave like anionic nanogels, i.e., they
undergo the volumetric change expected for PVCL, changing from the
swollen state to the collapsed state as the temperature increases (see
FIG. 3). This is because methyl methacrylate is pH sensitive and when the
pH of the medium exceeds the pKa of the carboxyl group from MMA
hydrolysis, said group ionizes providing negative charges to the nanogel
of the invention.

[0032] The concentration of VCL with respect to the total concentration of
the formulation (recipe) comprising the components necessary for
obtaining the polymer units interconnected with one another through a
cross-linking agent making up the polymer network of the biocompatible
nanogel of the invention can vary within a wide range; specifically,
between the concentration of VCL which does not form a macroscopic gel
when polymerized (maximum concentration) and the concentration of VCL
which allows obtaining the final desired diameter of the biocompatible
nanogel of the invention when polymerized (minimum concentration). In a
particular embodiment, the concentration of VCL is comprised between
0.5-3% by weight with respect to the total weight of the recipe. In an
even more particular embodiment, the concentration of VCL is
approximately 1% by weight with respect to the total weight of the
recipe.

Cross-Linking Agent

[0033] According to the present invention, the component "cross-linking
agent" is a difunctional monomer comprising at least two vinyl groups.

[0034] In a specific embodiment, said cross-linking agent is a dextran
with two or more vinyl groups.

[0035] In another specific embodiment, said cross-linking agent is
N,N'-methylenebisacrylamide (BA).

[0036] In another particular embodiment, said cross-linking agent is a
difunctional monomer which, in addition to two vinyl groups, comprises at
least one ethylene glycol unit. Non-limiting illustrative examples of
said cross-linking agent include a difunctional monomer of general
formula

##STR00002##

wherein

[0037] X is hydrogen or methyl, and

[0038] n is an integer comprised between 1 and 90.

[0039] In a specific embodiment, when "n" is 1 and "X" is hydrogen, the
cross-linking agent is ethylene glycol diacrylate (EGDA), and when "n" is
greater than 1 and "X" is hydrogen, the cross-linking agent is
poly(ethylene glycol diacrylate) (PEGDA).

[0040] In another specific embodiment, when "n" is 1 and "X" is methyl,
the cross-linking agent is ethylene glycol dimethacrylate (EGDMA), and
when "n" is greater than 1 and "X" is methyl, the cross-linking agent is
poly(ethylene glycol dimethacrylate) (PEGDMA).

[0041] In a particular embodiment, the cross-linking agent is a
difunctional monomer selected from the group consisting of a dextran with
two or more vinyl groups, N,N'-methylenebisacrylamide (BA), EGDA, PEGDA,
EGDMA, PEGDMA and mixtures thereof.

[0042] Some of said cross-linking agents have the characteristic where, in
addition to the oxygen atoms of the carbonyl groups, the oxygen atoms
present in the ethylene glycol units can form hydrogen bonds with
carboxyl groups.

[0043] The concentration of cross-linking agent with respect to the VCL
present in the formulation (recipe) comprising the components necessary
for obtaining the polymer units interconnected with one another through
said cross-linking agent making up the polymer network of the
biocompatible nanogel of the invention can vary within a wide range,
depending on the desired diameter of the biocompatible nanogel of the
invention (at temperatures higher or lower than the LCST); nevertheless,
in a particular embodiment, the concentration of cross-linking agent
present in said recipe is comprised between 2% and 10% by weight with
respect to VCL; in an even more particular embodiment, the concentration
of cross-linking agent present in said recipe is approximately 8% by
weight with respect to VCL.

Cationic Initiator

[0044] According to the present invention, the component "cationic
initiator" refers to a compound which decomposes by means of thermal
activation generating positively charged reactive species which start the
radical polymerization process. As a result, the initiator used in the
invention is a cationic thermal initiator.

[0045] The cationic nature of the initiator is of vital importance in
nanogel preparation given that it is the compound which will confer the
positive charge to the nanogel, the charge being what allows the nanogel
nanoparticles to undergo an instantaneous volumetric change in the phase
transition temperature and allows said change to involve the transition
from the collapsed state to the swollen state as the temperature
increases.

[0046] Although virtually any suitable initiator capable of decomposing
and generating positive species could be used, in a particular
embodiment, said initiator is selected from the group consisting of
2,2-azobis(N,N'-dimethylene isobutyramidine) dihydrochloride (ADIBA),
2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate,
2,2'-azobisisobutyramidine dihydrochloride (AIBA),
2,2'-azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane}
dihydrochloride and 2,2'-azobis(1-imino-1-pyrrolidino-2-ethylpropane)
dihydrochloride.

[0047] The respective formulas of these initiators are shown below:

##STR00003##

[0048] According to a preferred embodiment, the initiator used is
2,2-azobis(N,N'-dimethylene isobutyramidine) dihydrochloride (ADIBA),
which decomposes thermally providing reactive cationic species which
allow starting the polymerization and cross-linking of VCL via radical.

[0049] The concentration of initiator which can be used for putting said
method into practice can vary within a wide range; nevertheless, in a
particular embodiment, the concentration of initiator is comprised
between 0.3% and 1.5% by weight with respect to VCL; in even more
particular embodiments, the concentration of initiator present in said
recipe ranges between 0.3 and 1.2% by weight with respect to VCL.

Emulsifier

[0050] Emulsifier confers colloidal stability to the cationic nanogels of
the invention. It covers the polymer network making up the nanogel
nanoparticles. According to the present invention, the emulsifier can be
both cationic and non-ionic emulsifier.

[0051] Although virtually any suitable cationic or non-ionic emulsifier
could be used, in a particular embodiment, the emulsifier is a non-ionic
emulsifier, preferably Tween 20. The formula corresponding to this
compound is shown below:

##STR00004##

wherein a+b+c+d=20

[0052] In another particular embodiment, said emulsifier is a cationic
emulsifier. More particularly, said cationic emulsifier is an
alkyltrimethylammonium halide. The alkyltrimethylammonium halide is
preferably selected from hexadecyltrimethylammonium bromide (HDTAB) and
dodecyltrimethylammonium bromide (DTAB), respectively having the
following formulas:

##STR00005##

[0053] In a still more preferred embodiment, the cationic emulsifier is a
quaternary ammonium block copolymer, this being understood as a block
copolymer having a quaternary ammonium terminal group, such as for
example, a styrene and 2-(dimethylamino)ethyl methacrylate block
copolymer of formula:

##STR00006##

wherein n and m are integers comprised between 5 and 50.

[0054] Among the compounds corresponding to this formula, the use of the
styrene and 2-(dimethylamino)ethyl methacrylate block copolymer defined
in the preceding formula in which n=7 and m=13 is preferred. Throughout
this specification said compound is called S7A13.

[0055] These block copolymers can be synthesized by any method known by a
person skilled in the art. Nevertheless, in a particular embodiment, the
synthesis of said copolymer is carried out in three steps. A first step
of polymerizing the polystyrene chain which is carried out in the
presence of an initiator and is catalyzed by a copper complex, such as
for example CuBr2/tris[2-(dimethylamino)ethyl]amine; a second step
of elongating the polystyrene chain with 2-(dimethylamino) ethyl
methacrylate (also called DMAEMA) which is performed by reacting the
polystyrene with DMAEMA in the presence of the mentioned copper complex;
and a third step of quaternizing the amino group of methacrylate by
reacting with an alkyl halide, such as methyl iodide for example. A
detailed example of the synthesis of this block copolymer is detailed in
Example 1, wherein n=7 and m=13.

[0056] The concentration of emulsifier which can be used for putting the
method of the invention into practice can vary within a wide range;
nevertheless, in a particular embodiment, the concentration of emulsifier
is comprised between 0.2% and 25% by weight with respect to VCL; in an
even more particular embodiment, the concentration of emulsifier present
in said recipe can range between 0.3 and 20% by weight with respect to
VCL.

Obtaining the Cationic Nanogel Object of the Invention

[0057] The biocompatible cationic nanogel of the invention is obtained as
a result of cross-linking PVCL polymer units interconnected with one
another through the cross-linking agent, generating a polymer network.
Said polymer network can be obtained by means of a method of
polymerization of the main monomer (VCL) in the presence of said
cross-linking agent in a dispersed medium, such as emulsion
polymerization. In the context of the invention, the emulsion
polymerization of said monomers also comprises the use of a cationic
initiator and of a cationic or non-ionic emulsifier. The polymerization
of a monomer, as well as the copolymerization of two or more different
monomers in a dispersed (heterogeneous) medium is known by the persons
skilled in the art. In general, in such method of polymerization, water
(e.g., distilled water) is used as the continuous reaction medium.

[0058] The polymerization reaction can be carried out at a temperature
comprised within a wide range; nevertheless, it is very important to take
into account the phase change critical temperature (LCST) of the polymer
units making up the polymer network of the biocompatible nanogel of the
invention when choosing the reaction temperature. In a particular
embodiment, the polymerization reaction is carried out at a temperature
greater than the LCST of the different polymer units forming part of the
biocompatible nanogels of the invention. In a specific embodiment, the
polymerization reaction is carried out at a temperature equal to or
higher than 60° C.

[0059] Likewise, the stirring speed of the formulation (recipe) containing
the components necessary for generating the biocompatible nanogel of the
invention by means of polymerization in a dispersed medium as mentioned
above can vary within a wide range; nevertheless, the stirring speed must
be high enough in order to achieve a homogenous mixture of the dispersion
(reaction mixture) when synthesizing the biocompatible nanogels of the
invention. In a particular embodiment, the stirring speed is equal to or
greater than 200 rpm; in another particular embodiment, the stirring
speed is comprised between 200 and 400 rpm, for example approximately 300
rpm.

[0060] The polymerization reaction can be carried out in a reactor
discontinuously (or in batches) or semi-continuously.

[0061] The technical features of the biocompatible nanogels of the
invention will depend, among other factors, on the nature and
concentration of VCL used, cross-linking agent, emulsifier, initiator,
and reaction temperature. However, by way of non-limiting illustration,
the values of some technical features of said biocompatible nanogels of
the invention can be indicated in ranges:

[0062] Diameter of collapsed
particle (15° C.): 30-60 nm

[0063] Diameter of swollen particle
(55° C.): 150-800 nm

[0064] Phase transition temperature:
32.5° C.-38° C.

[0065] The cationic nanogels of the invention may both be dispersed in
water and lyophilized. Once lyophilized, if they are resuspended in an
aqueous medium, they maintain their properties and capacity to
swell/collapse with respect to temperature changes of the dispersion
medium.

Biocompatible Nanogels Loaded with a Biologically Active Agent

[0066] As mentioned above, the cationic biocompatible nanogels of the
invention are capable of uptake or encapsulating biologically active
agents. Therefore, in a particular embodiment, the biocompatible nanogel
of the invention further comprises a biologically active agent. In
general, said biologically active agent can be inside the biocompatible
nanogel of the invention; nevertheless, sometimes the biologically active
agent can also be bound to or adsorbed on the outer surface of said
nanogel.

[0067] The cationic biocompatible nanogels loaded with biologically active
agents provided by this invention are in the form of nanoparticles which
are sensitive to the temperature changes of the medium in which they are
dispersed and can swell and collapse (i.e., change in size), thus
responding to temperature stimuli. They particularly undergo an
instantaneous volumetric change in the phase temperature of the main
polymer making up the polymer network, changing from a collapsed state to
a swollen state as the temperature increases.

[0068] The biocompatible nanogel nanoparticles loaded or not loaded with a
biologically active agent provided by this invention can be lyophilized
and then resuspended in an aqueous medium, preserving their properties
and sensitivities.

[0069] The temperature sensitive biocompatible nanogels of the present
invention have a number of advantages with respect to other "intelligent"
materials which cannot be used in drug dosing because they do not meet
the biocompatibility condition. In these novel nanogels, the driving
force controlling the absorption and subsequent dosing of the
biologically active agent has a certain covalent character, which entails
an additional advantage both in carrying the biologically active agent
and in dosing it at the chosen site.

[0070] On the other hand, unlike the anionic nanogels of PVCL, the
cationic nanogels of the invention allow immediately releasing the active
ingredient due to the instantaneous volumetric change they undergo.

[0071] As used herein, the term "biologically active agent" refers to any
substance which is administered to a subject, preferably a human being,
for prophylactic or therapeutic purposes; i.e., any substance which can
be used in treating, healing, preventing or diagnosing a disease or for
improving the physical and mental well-being of humans and animals, for
example, drugs, antigens, allergens, etc.

[0072] The biocompatible nanogel of the invention or the biocompatible
nanogel nanoparticles of the invention can incorporate one or more
biologically active agents regardless of the solubility characteristics
thereof.

[0073] The chemical nature of the biologically active agent can vary
within a wide range from small molecules to macromolecular compounds
(peptides, polynucleotides, etc.). In a particular embodiment, said
biologically active agent is a peptide or a protein. In another
particular embodiment, said biologically active agent is a nucleoside, a
nucleotide, an oligonucleotide, a polynucleotide or a nucleic acid (e.g.,
RNA or DNA). In another particular embodiment, said biologically active
agent is a small (organic or inorganic) molecule; these molecules are
generally obtained by semi-synthetic or chemical synthesis methods, or
alternatively they are isolated from their sources. In a specific
embodiment, said small (organic or inorganic) molecule has a relatively
low molecular weight, generally equal to or less than 5,000, typically
equal to or less 2,500, advantageously equal to or less 1,500. Many
therapeutic active ingredients (drugs) have these characteristics and can
therefore be used for putting the present invention into practice.

[0075] Therefore, the nanogels provided by this invention are
biocompatible materials useful as carriers of biologically active agents,
capable of absorbing (encapsulating/uptake) both hydrophobic and
hydrophilic biologically active agents, and suitably dosing them since
they respond to the temperature stimuli of the medium in which they are
dispersed.

[0076] The biocompatible nanogels of the invention can uptake or
encapsulate the biologically active agent by conventional methods. In a
particular embodiment, the polymerization reaction in a dispersed medium
for producing the biocompatible nanogel of the invention can be carried
out in the presence of the biologically active agent for the purpose of
encapsulating said agent therein. Alternatively, the method for obtaining
the biocompatible nanogel loaded with a biologically active agent
provided by this invention comprises contacting a dispersion comprising a
biocompatible nanogel of the invention with a solution comprising said
biologically active agent (or agents) to be encapsulated.

[0077] More specifically, in a particular embodiment, a dispersion
comprising the biocompatible nanogels of the invention (or nanoparticles
based on said nanogels) is mixed with a solution of the biologically
active agent to be uptake, at a specific pH; the resulting solution is
stirred for a suitable time period and the nanogels of the invention
loaded with the biologically active agent are separated by conventional
methods, such as centrifugation (e.g., at 10,000-15,000 rpm and
20° C.). After separating the loaded nanogels the supernatant is
analyzed by conventional methods, e.g., by means of spectrophotometry.
The amount of biologically active agent uptake or absorbed can be
calculated by knowing the concentration of biologically active agent
which has been used and the concentration of biologically active agent
which remained in the supernatant.

Pharmaceutical Compositions

[0078] In another aspect, the invention relates to a pharmaceutical
composition comprising a biocompatible nanogel loaded with a biologically
active agent provided by this invention and a pharmaceutically acceptable
excipient, vehicle or adjuvant.

[0079] Non-limiting illustrative examples of pharmaceutical compositions
include any composition (solid or semi-solid) intended for oral, buccal,
sublingual, topical, ocular, intranasal, pulmonary, rectal, vaginal,
parenteral, topical administration, etc. In a particular embodiment, the
pharmaceutical composition is administered orally due to its
biocompatibility. The biocompatible nanogels of the invention are
"intelligent" materials which provide a more controlled release of the
biologically active agent and protect said biologically active agents
during release, their bioavailability thus being able to be controlled in
a uniform and constant manner. The reversibility of the swelling
properties of these nanogels makes these materials the excellent vehicles
for carrying both small biologically active agents and new macromolecular
drugs (for example, peptides) and other therapeutic products.

[0080] The pharmaceutical compositions described will comprise the
excipients suitable for each formulation. In the case of oral
formulations in the form of tablets or capsules, for example, binding
agents, disintegrants, lubricants, loading agents, enteric coating, etc.,
will be included if necessary. Solid oral formulations are prepared in a
conventional manner by mixing, dry or wet granulation and by
incorporating the biocompatible nanogel of the invention loaded with the
biologically active agent. The pharmaceutical compositions can also be
adapted for parenteral administration, for example in the form of sterile
solutions, suspensions or lyophilized products, in the suitable dosage
form; in this case, said pharmaceutical compositions will include the
suitable excipients, such as buffers, surfactants, etc. In any case, the
excipients will be chosen depending on the pharmaceutical dosage form
selected. A review of the different pharmaceutical dosage forms of drugs
and of their preparation can be found in the book "Tratado de Farmacia
Galenica", by C. Fauli i Trillo, 10 Edition, 1993, Luzan 5, S.A. de
Ediciones.

[0081] The proportion of the biologically active agent incorporated in the
biocompatible nanogels of the invention can vary within a wide range, for
example it can be up to 50% by weight with respect to the total weight of
the nanoparticles. Nevertheless, in each case the suitable proportion
will depend on the biologically active molecules incorporated.

[0082] The invention is described below by means of several illustrative
non-limiting examples of the invention.

[0085] The following materials were used in this ARGET ATRP synthesis:

[0086] Styrene (Aldrich) which was purified by means of distillation (5
mbar at 25° C.);

[0087] Dimethylethylamine methacrylate (Aldrich)
which was also purified by means of distillation (2 mbar at 25°
C.)

[0088] Copper(II) bromide (CuBr2) (Aldrich) which was used as it
was acquired (99-100% of purity).

[0089] Ethyl-2-bromoisobutyrate (EBiB)
(Aldrich) which was purified by means of distillation (3 mbar at
30° C.); and

[0090] tris[2-(dimethylamino)ethyl]amine
(Me6TREN) which was prepared as described in Macromolecules, 1998,
31, 5958 or in Inorg. Chem., 1966, 5, 41.

Synthesis of the Macroinitiator PSty

[0091] The macroinitiator PSty was prepared using ethyl 2-bromoisobutyrate
(EBiB) as an initiator. The reaction was catalyzed by the
CuBr2/tris[2-(dimethylamino)ethyl]amine (Me6TREN) complex under
argon. Anisole was used as solvent and ascorbic acid was added in excess
as reducing agent for reducing Cu(II) to Cu(I). The different components
used in the synthesis of the macroinitiator PSty as well as the amounts
of each of them are summarized in Table 1.

[0092] The ligand (Me6TREN) and CuBr2 were mixed in a Schlenk
tube with 5 grams of anisole to form the
CuBr2/tris[2-(dimethylamino)ethyl]amine complex. Argon was bubbled
through the solution of the complex for 15 minutes while it was stirred.
The ascorbic acid was dissolved in the remaining anisole and was added to
a 250 mL three-neck flask. The styrene and the solution with the complex
were transferred to the flask. 3 cooling cycles were performed on the
reaction mixture and it was kept under argon atmosphere throughout the
entire reaction. The temperature of the oil bath was set to 90° C.
and when the temperature of the reaction mixture was 90° C., the
initiator EBiB was added for the purpose of starting polymerization. The
reaction proceeded for 32 hours. 1 mL aliquots were taken during the
reaction. The molecular weights were measured by means of size exclusion
chromatography (SEC). The conversion was followed by means of gas
chromatography using the reaction solvent as the internal standard. The
final product was purified by means of precipitation in methanol. The
polymer obtained was vacuum dried in an oven at 40° C. for 24
hours. The product was characterized by means of SEC and MALDI-TOF and
1H NMR.

[0093] The polymerization was suitably controlled, providing polymers with
controlled molecular weights and low polydispersities. The final
conversion was 80%. According to the SEC analysis, the average molecular
weight in number (Mn) was 878 g/mol with a polydispersity of 1.2.
MALDI-TOF analyses confirmed these results. The maximum theoretical
content of copper in the polymer before purification was approximately
600 ppm. This content is very low if compared with the values for normal
ATRP in which this content is around 40,000 ppm.

Elongation of the Chain of the Macroinitiator PSty-Br with DMAEMA

[0094] The chain of the macroinitiator (PSty-Br) was elongated with DMAEMA
for the purpose of providing a block copolymer. The macroinitiator
(PSty-Br), DMAEMA and 95% by weight of anisole were added in a three-neck
flask. 3 cooling cycles were applied to the reaction mixture and it was
kept under argon atmosphere throughout the entire reaction. The reaction
mixture was heated at 25° C. On the other hand, a mixture of the
CuBr2/tris[2-(dimethylamino)ethyl]amine (Me6TREN) complex and
5% of anisole was degassed with argon for 15 minutes and was added to the
previous reaction mixture. The components used for the chain elongation
reaction of PSty-Br with DMAEMA, as well as their corresponding amounts,
are shown in Table 2. The reaction proceeded for 24 hours. The final
product was recrystallized in n-heptane to remove the PSty-Br which had
not reacted. Even when the final product contained very small amounts of
copper, the block copolymer was ultra-purified by means of dissolving 50
g of the polymer obtained in 1 L of water acidified with 10 mL of
HClaq (33%). The solution was then neutralized by means of adding 20
g of a 25% NaOH solution in water. The precipitated block copolymer was
recovered and then vacuum dried in an oven at 40° C. for 24 hours.
The composition of the block copolymer was determined by means of 1H
NMR.

[0095] The chain elongation reaction showed a linear behavior when Mn
was represented with respect to the conversion, which shows that the
reaction proceeds under a controlled mechanism. The final conversion was
90% after 24 hours. Between 10 and 20% of the total amount of the
macroinitiator PSty initially incorporated was recovered after the end of
the reaction, showing that a significant fraction of the macroinitiator
cannot be elongated. The final composition of the block copolymer
according to the 1H NMR spectrum comprised 13 units of DMAEMA and 7
units of styrene. After the final purification, it was confirmed by
atomic spectroscopy that the total content of copper was virtually
negligible.

Quaternization of PSty7-b-PDMAEMA13

[0096] The block copolymer was dissolved by 4.5 times its weight in THF
and CH3I was added in two-fold molar excess. The reaction proceeded
for 24 hours at room temperature. Polystyrene-b-poly(trimethyl ammonium
ethyl methacrylate) (PSty-b-PTMAEMA) was recovered from the mixture after
completing the reaction by means of removing the THF and subsequently
washing the polymer with diethylether. The excess CH3I used assured
that the total amount of the DMAEMA groups was completely quaternized.
1H NMR analyses were performed using D2O as a solvent to show
that all the amino groups were quaternized.

Example 2

Synthesis of Cationic Nanogels Stabilized with S7A13

[0097] These nanogels were synthesized in batches (discontinuously) in a
reactor by means of emulsion copolymerization. VCL was used as the main
monomer, PEGDA as the cross-linking agent, S7A13 as the emulsifier and
ADIBA as the initiator.

[0098] The nanogel particles were prepared using different concentrations
of emulsifier and initiator. Tables 3 and 4 show the recipes and reaction
conditions used in the production of these nanogels varying the
concentration of emulsifier and initiator, respectively.

Synthesis of Cationic Nanogels Stabilized with a Different Type of
Emulsifier (S7A13, HDTAB, DTAB, or Tween 20)

[0099] These nanogels were synthesized in batches or discontinuously in a
reactor by means of emulsion copolymerization. VCL was used as the main
monomer, PEGDA as the cross-linking agent and ADIBA as the initiator. The
nanogel particles were prepared using different emulsifiers: S7A13,
HDTAB, DTAB, or Tween 20.

[0100] Table 5 shows the recipes and reaction conditions used in the
production of these nanogels.

Effect of Temperature of the Medium on the Average Hydrodynamic Diameter
of the Nanogel

[0101] FIG. 4 shows the response of the cationic nanogels, synthesized
with different concentrations of S7A13, to the temperature changes of the
dispersion medium. As can be seen, the nanogels are collapsed when the
temperature of the medium is between 10-30° C. An instantaneous
change or leap in size (average hydrodynamic diameter) then occurs which
coincides with the phase transition temperature of the
polyvinylcaprolactam (PVCL) homopolymer which would be obtained upon
polymerizing the vinyl monomer (VCL) used in the synthesis as the main
monomer alone. At low temperatures, sizes do not vary with the
concentration of emulsifier. However, differences are observed when the
temperature of the medium is above the transition temperature: the higher
the concentration of emulsifier the larger the size of the nanogel.

[0102] Next, FIG. 5 shows the response of the cationic nanogels,
synthesized by varying the concentration of initiator, to the temperature
changes of the medium. It can be seen that the reduction of the
concentration of initiator causes the reduction of the size of the
nanogels at high temperatures. Furthermore, the behavior of the nanogel
is reversible and the leap occurs both upon heating and upon cooling the
medium in which the nanogel is dispersed.

[0103] In terms of the effect of the type of emulsifier, FIG. 6 show that
the nanogels synthesized with both cationic emulsifier (HDTAB or DTAB)
and non-ionic emulsifier (Tween 20) have the same behavior as nanogel E20
in which the amphiphilic block copolymer S7A13 has been used. It must be
taken into account that even though a cationic emulsifier has not been
used in the synthesis of nanogel NI20, it is also a cationic nanogel due
to the positive charge from the initiator (ADIBA).

[0104] By comparing the diameters obtained with those of nanogel E20 used
as a reference, it is observed that there are differences at both low and
high temperatures. The nanogels synthesized using HDTAB or DTAB have the
largest collapsed diameter (110 nm). The size of nanogel NI20 is 90 nm in
the collapsed state, whereas the size of nanogel E20 is smaller, 50 nm.

[0105] In terms of the leap in the transition, it decreases when HDTAB or
DTAB is used as emulsifier, swollen sizes less than those of nanogels E20
and NI20 being obtained. Therefore, the improvement provided by the use
of the amphiphilic block copolymer S7A13 in the recipe with respect to
the instantaneous leap from the collapsed state to the swollen state
experienced by the nanogel particles as the temperature increases above
the phase transition value of PVCL is evident.

[0106] It is still found that the behavior of the nanogel is reversible
because the instantaneous leap from the collapsed state to the swollen
state occurs both upon heating and upon cooling the medium in which the
nanogel is dispersed.

Example 5

Lyophilization-Redispersion

[0107] FIG. 7 shows the behavior of the reference cationic nanogel (E20)
in dispersion (non-lyophilized) and after its lyophilization and
subsequent redispersion (resuspension time in minutes). The behavior of
the nanogel lyophilized and then resuspended in water is the same as that
of the non-lyophilized nanogel, i.e., collapsed at low temperatures and
swollen at high temperatures. It is observed that the instantaneous leap
occurs at the same temperature and that the collapsed sizes at low
temperatures (10-30° C.) remain identical, whereas the sizes
corresponding to the swollen state at high temperatures are affected by
lyophilization, being smaller than those the of the nanogel which has not
been lyophilized.